The immune system serves a vital role in protecting the body against infectious agents. It is well established, however, that a number of disease states and/or disorders are a result of either abnormal or undesirable activation of immune responses. Common examples include graft versus host disease (GVHD), graft rejection, inflammation, and autoimmune linked diseases such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), and certain forms of arthritis.
In general, an immune response is activated as a result of either tissue injury or infection. Both cases involve the recruitment and activation of a number of immune system effector cells (i.e. B- and T-lymphocytes, macrophages, eosinophils, neutrophils) in a process coordinated through a series of complex cell-cell interactions. A typical scenario by which an immune response is mounted against a foreign protein is as follows: Foreign proteins captured by antigen presenting cells (APC's) such as macrophages or dendritic cells are processed and displayed on the cell surface of the APC. Circulating T-helper cells which express an immunoglobulin that recognizes (i.e. binds) the displayed antigen undergo activation by the APC. These activated T-helpers in turn activate appropriate B-cell clones to proliferate and differentiate into plasma cells that produce and secrete humoral antibodies targeted against the foreign antigen. The secreted humoral antibodies are free to circulate and bind to any cells expressing the foreign protein on their cell surface, in effect marking the cell for destruction by other immune effector cells. In each of the stages described above, direct cell-cell contact between the involved cell types is required in order for activation to occur [Gruss et al., Leuk. Lymphoma, 24, 393 (1997)]. In recent years, a number of cell surface receptors that mediate these cell-cell contact dependent activation events have been identified. Among these cell surface receptors is CD40 and its physiological ligand, CD40 Ligand (CD40L).
CD40 was first characterized as a receptor expressed on B-lymphocytes. It was later found that engagement of B-cell CD40 with CD40L expressed on activated T-cells is essential for T-cell dependent B-cell activation (i.e. proliferation, immunoglobulin secretion, and class switching. It was subsequently revealed that functional CD40 is expressed on a variety of cell types other than B-cells, including macrophages, dendritic cells, thymic epithelial cells, Langerhans cells, and endothelial cells. These studies have led to the current belief that CD40 plays a broad role in immune regulation by mediating interactions of T-cells with B-cells as well as other cell types. In support of this notion, it has been shown that stimulation of CD40 in macrophages and dendritic results is required for T-cell activation during antigen presentation [Gruss et al., Leuk. Lymphoma, 24, 393 (1997)]. Recent evidence points to a role for CD40 in tissue inflammation as well. Production of the inflammatory mediators IL-12 and nitric oxide by macrophages have been shown to be CD40 dependent [Buhlmann and Noelle, J. Clin. Immunol., 16, 83 (1996)]. In endothelial cells, stimulation of CD40 by CD40L has been found to induce surface expression of E-selectin, ICAM-1, and VCAM-1, promoting adhesion of leukocytes to sites of inflammation [Buhlmann and Noelle, J. Clin. Immunol., 16, 83 (1996); Gruss et al., Leuk. Lymphoma, 24, 393 (1997)]. Finally, a number of reports have documented overexpression of CD40 in epithelial and hematopoietic tumors as well as tumor infiltrating endothelial cells, indicating that CD40 may play a role in tumor growth and/or angiogenesis as well [Gruss et al., Leuk. Lymphoma, 24, 393 (1997); Kluth et al., Cancer Res., 57, 891 (1997)].
Due to the pivotal role that CD40 plays in humoral immunity, the potential exists that therapeutic strategies aimed at downregulating CD40 or interfering with CD40 signaling may provide a novel class of agents useful in treating a number of immune associated disorders, including but not limited to graft-versus-host disease (GVHD), graft rejection, and autoimmune diseases such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), and certain forms of arthritis. Inhibitors of CD40 may also prove useful as anti-inflammatory compounds, and could therefore be useful as treatment for a variety of inflammatory and allergic conditions such as asthma, rheumatoid arthritis, allograft rejections, inflammatory bowel disease, autoimmune encephalomyelitis, thyroiditis, various dermatological conditions, and psoriasis. Recently, both CD40 and CD154 have been shown to be expressed on vascular endothelial cells, vascular smooth muscle cells and macrophages present in atherosclerotic plaques, suggesting that inflammation and immunity contribute to the atherogenic process. That this process involves CD40 signaling is suggested by several studies in mouse models in which disruption of CD154 (by knockout or by monoclonal antibody) reduced the progression or size of atherosclerotic lesions. Mach et al., 1998, Nature, 394, 200-3, Lutgens et al., 1999, Nat. Med. 5, 1313-6.
Finally, as more is learned of the association between CD40 overexpression and tumor growth, inhibitors of CD40 may prove useful as anti-tumor agents and inhibitors of other hyperproliferative conditions as well.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of CD40. To date, strategies aimed at inhibiting CD40 function have involved the use of a variety of agents that disrupt CD40/CD40L binding. These include monoclonal antibodies directed against either CD40 or CD40L, soluble forms of CD40, and synthetic peptides derived from a second CD40 binding protein, A20. The use of neutralizing antibodies against CD40 and/or CD40L in animal models has provided evidence that inhibition of CD40 signaling would have therapeutic benefit for GVHD, allograft rejection, rheumatoid arthritis, SLE, MS, and B-cell lymphoma [Buhlmann and Noelle, J. Clin. Immunol, 16, 83 (1996)]. Clinical investigations were initiated using CD154 monoclonal antibody in patients with lupus nephritis. However, studies were terminated due to the development of thrombotic events. Boumpas et al., 2003, Arthritis Rheum. March; 48, 719-27.
Due to the problems associated with the use of large proteins as therapeutic agents, there is a long-felt need for additional agents capable of effectively inhibiting CD40 function. Antisense oligonucleotides avoid many of the pitfalls of current agents used to block CD40/CD40L interactions and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic and research applications. U.S. Pat. No. 6,197,584 (Bennett and Cowsert) discloses antisense compounds targeted to CD40.
Peptide nucleic acids, alternately referenced as PNAs, are known to be useful as oligonucleotide mimetics. In PNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units of oligonucleotides are replaced with novel groups. The sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. The base units, i.e., nucleobases, are maintained for hybridization with an appropriate nucleic acid target compound.
PNAs have been shown to have excellent hybridization properties as well as other properties useful for diagnostics, therapeutics and as research reagents. They are particularly useful as antisense reagents. Other uses include monitoring telomere length, screening for genetic mutations and for affinity capture of nucleic acids. As antisense reagents they can be used for transcriptional and translational blocking of genes and to effect alternate splicing. Further they can be used to bind to double stranded nucleic acids. Each of these uses are known and have been published in either the scientific or patent literature.
The synthesis of and use of PNAs has been extensively described. Representative United States patents that teach the preparation of and use of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,5539,083; 5,641,625; 5,714,331; 5,719,262; 5,766,855; 5,773,571; 5,786,461; 5,831,014; 5,864,010; 5,986,053; 6,201,103; 6,204,326; 6,210,892; 6,228,982; 6,350,853; 6,414,112; 6,441,130; and 6,451,968, each of which is herein incorporated by reference. Additionally PNA compounds are described in numerous published PCT patent applications including WO 92/20702. Further teaching of PNA compounds can be found in scientific publications. The first such publication was Nielsen et al., Science, 1991, 254, 1497-1500.
Depending on sequence, the solubility of PNAs can differ and, as such, some PNA sequences are not soluble as might be desirable for a particular use. It was suggested in Karras, et al., Biochemistry, 2001, 40, 7853-7859, that PNAs could mediate splicing activity in cells. They compared a PNA 15mer (a PNA having 15 monomeric units) to the same PNA having a single lysine amino acid jointed to its C terminus. They suggested that the attached, i.e., conjugated, lysine residue might improve the cellular uptake. However, they concluded that their present data “do not show a clear difference in activity between the PNA 15mer with and without a C-terminal lysine.”
In published application US-2002-0049173-A1, published Apr. 25, 2002, it was suggested that antisense compounds might have one or more cationic tails, preferable positively charged amino acids such as lysine or arginine, conjugated thereto. It was further suggested that one or more lysine or arginine residues might be conjugated to the C-terminal end of a PNA compound. No discrimination was made between the effects resulting from the conjugation of one lysine or arginine versus more than one of these lysine or arginine residues.
U.S. Pat. No. 6,593,292 suggests using guanidine or amidine moieties for uptake of various compounds including macromolecules. PNA is a suggested macromolecule. In one instance this patent suggests that the guanidine or amidine moieties comprise non-peptide backbones but in a further instance it suggested that the guanidine moiety will exist as a polyarginine molecule. However, no data is shown wherein any of these moieties are actually conjugated to a macromolecule and uptake is achieved.
In a transgenic mouse model, a 4-lysine conjugated PNA targeted to β-globin was demonstrated to provide efficacy in a range of tissues (Sazani et al., 2002, Nature Biotech. 20, 1228-1233).